CN114657091A

CN114657091A - Campylobacter enrichment culture solution and preparation method and application thereof

Info

Publication number: CN114657091A
Application number: CN202210204186.7A
Authority: CN
Inventors: 张茂俊; 顾一心; 葛安山; 杜庆宝
Original assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Current assignee: Qingdao Zhongchuangyike Biotechnology Co ltd; National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-06-24
Anticipated expiration: 2042-03-03
Also published as: CN114657091B

Abstract

The application is applicable to the technical field of biology and provides a campylobacter enrichment culture solution as well as a preparation method and application thereof, wherein the campylobacter enrichment culture solution comprises the following raw materials in parts by weight: 10-15 parts of brain extract, 6-7 parts of bovine brain heart extract, 8-12 parts of peptone, 1-3 parts of glucose, 6.5-12.5 parts of metal salt, 50 parts of blood, 0.001-0.002 part of nicotinic acid, 0.5-1.5 parts of deoxysodium cholate, 0.0001-0.0005 part of tween and 0.05-0.1 part of antibiotic. The method has the advantages that the components of the culture solution are scientifically optimized, the growth speed of the microaerophilic campylobacter is remarkably increased, and the culture and single bacterial colony of the campylobacter can be obtained in the detection of samples polluted by both campylobacter and other bacteria; in addition, this application is toPlease improve the mobility of the trophism live bacteria of campylobacter, and greatly improve the detection limit of the campylobacter in the hollow sample to 10⁰CFU。

Description

Campylobacter enrichment culture solution and preparation method and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to a campylobacter enrichment culture solution, and a preparation method and application thereof.

Background

Campylobacter jejuni and campylobacter coli are important food-borne pathogenic bacteria, and in developed countries such as europe and america, campylobacter jejuni and campylobacter coli are the first pathogenic bacteria to cause food poisoning. Infection by campylobacter jejuni is usually 100 times higher in developing countries than in developed countries.

Gastrointestinal tract diseases caused by campylobacter jejuni infection are called campylobacteriosis. The main pathogeny of campylobacter coli disease is campylobacter jejuni, accounting for more than 80%, campylobacter coli accounting for about 15% -20%, and the rest is campylobacter strains except for campylobacter jejuni and campylobacter coli. In addition to gastroenteritis, infection with campylobacter jejuni can lead to guillain-barre syndrome (GBS). Detection and monitoring of campylobacter jejuni is the key point for prevention and control of infection and diagnosis and treatment. Isolation and culture of bacteria is the gold standard for determining infection or contamination by pathogenic bacteria.

Campylobacter jejuni and Campylobacter coli belong to gram-negative bacteria, and have harsh in vitro culture nutritional conditions, and belong to fastidious bacteria. The prior method for isolated culture of campylobacter jejuni and campylobacter jejuni comprises direct culture and enrichment culture. Because the in vitro growth conditions are harsh, enrichment culture is an effective culture method. The enrichment fluid of the campylobacter commonly used at present mainly comprises Bolton enrichment fluid and Preston enrichment fluid. These two types of enrichment fluids are mainly prepared by adding growth-promoting lysed horse blood or sheep blood and antibiotics to a basic liquid culture medium (including meat peptone, albumin hydrolysate, yeast extract, etc.). The difference between the two enrichment solutions is mainly represented by the difference of antibiotic types: the antibiotics of the Bolton enrichment broth mainly comprise 4 types of cefoperazone sodium, vancomycin, trimethoprim and amphotericin B or cycloheximide; the Preston enrichment broth is mainly characterized in that two antibiotics, namely cefoperazone sodium and vancomycin, in the Bolton enrichment broth are replaced by polymyxin B and rifampicin. At present, the two kinds of enrichment liquids can not well promote the multiplication of campylobacter and inhibit the growth of other bacteria of the campylobacter. After the two types of enrichment liquids are used for enrichment, even if the selective solid culture medium is used for culture in the later period, campylobacter is difficult to separate from food samples, particularly polluted poultry meat, milk eggs and diarrhea patients.

Disclosure of Invention

The embodiment of the application aims to provide a campylobacter enrichment culture solution, and aims to solve the problems that the existing enrichment culture solution cannot well promote the proliferation of campylobacter and inhibit the growth of other campylobacter outside, and the detection sensitivity and specificity are low.

The embodiment of the application is realized in such a way that the campylobacter enrichment culture solution comprises the following raw materials in parts by weight:

10-15 parts of brain extract, 6-7 parts of bovine brain heart extract, 8-12 parts of peptone, 1-3 parts of glucose, 6.5-12.5 parts of metal salt, 50 parts of blood, 0.001-0.002 part of nicotinic acid, 0.5-1.5 parts of sodium deoxycholate, 0.0001-0.0005 part of tween and 0.05-0.1 part of antibiotic.

Another objective of the embodiments of the present application is to provide a method for preparing a campylobacter enrichment culture solution, comprising:

weighing the raw materials according to the formula of the campylobacter enrichment culture solution for later use;

dissolving brain extract, medulla bovis Seu Bubali extract, peptone, glucose, metal salt, nicotinic acid, sodium deoxycholate and tween in deionized water, and high-pressure treating to obtain mixed liquid;

and adding blood and antibiotics into the mixture, and uniformly mixing to prepare the campylobacter bacteria enrichment culture solution.

Another object of the embodiments of the present application is to provide an application of the campylobacter enrichment culture solution in campylobacter detection.

The campylobacter enrichment culture solution provided by the embodiment of the application can obviously improve the growth speed of microaerophilic campylobacter by scientifically optimizing the components of the culture solution, and can obtain the culture and single colony of the campylobacter in the detection of a sample polluted by both the campylobacter and other bacteria; the application improves the motion capability of the 'viable bacteria' of the campylobacter acidophilus, greatly improves the detection limit of the campylobacter in the hollow and colon samples, and is 10 times of the original limit⁴-10⁷CFU to 10⁰A CFU; meanwhile, only a small amount of enrichment culture solution is needed in the use process, and the using amount of the enrichment culture solution is far less than that of the existing enrichment culture solution.

Drawings

FIG. 1 is a graph showing the difference in growth of Campylobacter in the ICDC-CAMPY enrichment broth base combination and the Bolton enrichment broth base combination provided by the embodiment of the present application;

FIG. 2 is a bar graph showing the growth influence of different components of the enrichment fluid on campylobacter in the embodiment of the present application;

FIG. 3 is a graph showing the proliferation characteristics of ICDC-CAMPY enrichment liquid combined campylobacter at different times according to the embodiment of the present application;

FIG. 4 is a schematic diagram of counting the number of viable campylobacter (left) and infectious microbe (right) provided in the embodiment of the present application;

FIG. 5 is a schematic representation of results of Bolton (Preston) enrichment broth culture of a contaminated poultry sample provided in an example of the present application;

FIG. 6 is a schematic diagram of the results of the ICDC-CAMPY enrichment culture of a contaminated poultry sample provided in the examples of the present application;

FIG. 7 is a schematic diagram of the results of the Bolton (Preston) enrichment culture of a contaminated milk sample according to the present embodiment;

FIG. 8 is a schematic diagram of the enrichment culture result of a contaminated milk sample ICDC-CAMPY provided by the embodiment of the application;

FIG. 9 is a schematic diagram of the culture result of contaminated egg white Bolton (Preston) enrichment broth provided by the embodiment of the present application;

FIG. 10 is a schematic diagram of the enrichment culture result of a contaminated egg white sample ICDC-CAMPY provided by the embodiment of the application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

The currently available enrichment solutions of Bolton and Preston, which need enrichment and are combined with a solid culture medium containing selective antibiotics, can partially separate campylobacter, but the detection sensitivity and specificity are significantly lower than those of the enrichment culture solution of campylobacter provided by the embodiment of the application. Especially, when the pollution to the mixed bacteria in the sample (or specimen) is high, the nutrient components of the culture medium of the enrichment solution are not enough to promote the growth of the campylobacter, and the slow growth of the campylobacter, the fast growth of the mixed bacteria and the poor inhibition of the inhibitor can not be realized, so that the culture detection rate is low or the campylobacter cannot be detected. By applying the method, the campylobacter can not be detected in food samples in most regions and specimens of diarrhea patients in food safety monitoring and pathogen monitoring of infectious diarrhea of China.

In view of the current situation, the embodiment of the application provides a novel high-efficiency bacteria-increasing liquid and bacteria-increasing culture method for campylobacter coli in empty and colon, and the detection rate of campylobacter in samples and specimens in China is remarkably improved.

Specifically, the embodiment of the application provides a campylobacter enrichment culture solution (named as ICDC-CAMPY), which comprises the following raw materials in parts by weight:

The ICDC-CAMPY optimizes the composition of the existing enrichment liquid, modifies the components of a basic culture medium, removes nitrogen sources and carbon sources such as meat peptone, albumin and hydrolysate of complex protein and yeast extract in the general basic culture medium based on Bloton enrichment liquid, and changes the nitrogen sources and the carbon sources into the solid bovine brain core extract, peptone, glucose and nicotinic acid which have the promotion effect on fastidious bacteria, so that the growth speed of microaerophilic campylobacter is obviously improved, and the culture and single colony of the campylobacter can be obtained in the detection of a sample simultaneously polluted by the campylobacter and other bacteria.

The research of the application determines the minimum content of the raw materials, such as brain extract not less than 10 parts, bovine brain extract not less than 6 parts, peptone not less than 8 parts, glucose not less than 1 part, metal salt not less than 6.5 parts, blood not less than 50 parts, nicotinic acid not less than 0.001 part, deoxysodium cholate not less than 0.5 part, tween not less than 0.0001 part and antibiotic not less than 0.05 part; any material content below the minimum level will result in a decrease in the sensitivity or detection limit of the assay. In addition, it will be appreciated by those skilled in the art that while the use of a minimum or suitable amount is appropriate to achieve equivalent results, the use of an appropriate amount of increase within this range of amounts to achieve the same technical result is intended to fall within the scope of the present application.

In addition, the ICDC-CAMPY promotes the growth of campylobacter and inhibits the pollution of other mixed bacteria by adding a culture inhibitor and a surfactant (sodium deoxycholate and Tween), and the effect of the enrichment solution on standing is not obviously different from that of vibration enrichment.

In the embodiment of the application, the metal salt comprises the following raw materials in parts by weight:

4-6 parts of sodium chloride, 2-5 parts of disodium hydrogen phosphate, 0.25-0.5 part of sodium pyruvate, 0.25-0.5 part of sodium metabisulfite and 0.15-0.3 part of ferrous sulfate.

Compared with the commonly used enrichment solution, the ICDC-CAMPY increases the application concentrations of sodium pyruvate, sodium metabisulfite and ferrous sulfate, remarkably promotes the growth of campylobacter and inhibits the growth of other mixed bacteria; wherein, the concentration of the disodium hydrogen phosphate is increased to 2-5g/L, the concentration of the sodium pyruvate and the sodium metabisulfite is increased to 0.5-0.75g/L from the original 0.25g/L, and the concentration of the ferrous sulfate is increased to 0.15-0.3 g/L; further, it is preferable that the concentration of disodium hydrogenphosphate is adjusted to 3.5g/L, the concentrations of sodium pyruvate and sodium metabisulfite are adjusted to 0.625g/L, the concentration of ferrous sulfate is adjusted to 0.225g/L, and the concentration of disodium hydrogenphosphate is adjusted to 3.5g/L to promote the growth of Campylobacter and to suppress the growth of other bacteria.

In the embodiment of the application, the blood is one of defibrinated sheep blood, rabbit blood and fetal calf serum.

In the embodiment of the application, the antibiotic comprises the following raw materials in parts by weight:

0.01-0.02 part of vancomycin, 0.01-0.015 part of amphotericin B, 0.01-0.012 part of rifampicin, 0.01-0.015 part of Trimethoprim (TMP) and 0.02-0.032 part of cefoperazone sodium.

Aiming at the pollution characteristics of Chinese samples, the application verifies that the combination of five antibiotics with lower application concentrations, namely vancomycin, amphotericin B, rifampicin, TMP and cefoperazone sodium, can play a role in inhibiting mixed bacteria, and improves the selectivity and specificity of the campylobacter enrichment culture solution on the basis of improving the growth speed of the campylobacter.

The embodiment of the application also provides a preparation method of the campylobacter enrichment culture solution, which comprises the following steps:

step S1: weighing the raw materials according to the formula of the campylobacter enrichment culture solution for later use.

Step S2: dissolving brain extract, medulla bovis Seu Bubali extract, peptone, glucose, metal salt, nicotinic acid, sodium deoxycholate, and tween in deionized water, and high-pressure treating to obtain mixed liquid.

Optionally, the step S2 includes:

dissolving brain extract, medulla bovis Seu Bubali extract, peptone, glucose, metal salt, nicotinic acid, sodium deoxycholate and tween in 1000 parts of deionized water, and high-pressure treating at 121 deg.C for 15min to obtain mixed liquid.

Step S3: and adding blood and antibiotics into the mixture, and uniformly mixing to prepare the campylobacter bacteria enrichment culture solution.

Optionally, the step S3 includes:

and when the mixed liquid is cooled to 45-55 ℃, adding blood and antibiotics for uniform mixing to prepare the campylobacter bacteria enrichment culture solution.

The embodiment of the application also provides application of the campylobacter enrichment culture solution in campylobacter detection.

It is noted that with the ICDC-CAMP enrichment broth of the present application, only 4mL of enrichment broth per sample was required for enrichment to obtain good culture results (see the example study on samples below), but according to the previous national standard and other general methods, at least 225mL of Bolton or Preston enrichment broth was required for 25g of poultry, and the results were poor. The campylobacter enrichment culture solution has the detection limit of 10 in poultry meat⁰CFU, the contamination of extremely low amounts of Campylobacter in the sample can be detected; in addition, in the field of pollutionThe detection limit of the milk and the egg is less than 10¹The detection limit of CFU and other enriched liquid is more than 10⁴CFU even requires 10⁷CFU can be detected.

Examples of certain embodiments of the present application are given below, which are not intended to limit the scope of the present application.

In addition, it should be noted that the numerical values given in the following examples are as precise as possible, but those skilled in the art will understand that each numerical value should be understood as a divisor rather than an absolutely exact numerical value due to measurement errors and experimental operational problems that cannot be avoided.

Example 1 (differential analysis of the changes in the basal culture Components of the ICDC-CAMPY enrichment broth compared with the conventional enrichment broth for Campylobacter)

ICDC-CAMPY enrichment liquid basic combination: 12.5 parts of solid brain extract, 6.5 parts of bovine brain heart extract, 10 parts of peptone, 2 parts of glucose and 0.001 part of nicotinic acid.

Basic combination of Bolton enrichment liquid: 10 parts of meat peptone, 5 parts of a complex protein hydrolysate and 5 parts of a yeast extract.

And (3) measuring a growth curve:

(1) carrying out passage on the campylobacter standard strain NCTC11168 in a Karmali culture medium, and carrying out microaerophilic culture at 37 ℃ for 24 h;

(2) collecting the bacteria with vigorous growth, suspending the bacteria in 5mL of physiological saline, and adjusting the OD of the bacteria solution₆₀₀Is 1.0;

(3) respectively diluting the bacteria liquid by 20 times by using ICDC-CAMPY enrichment liquid and Bolton enrichment liquid culture medium, wherein OD is OD₆₀₀Carrying out microaerophilic culture at the speed of 0.05 and at the speed of 180 rpm/min;

(4) the OD of the bacterial liquid was measured every 3h (average value of 3 samples at each time point), and pure ICDC-CAMPY enrichment liquid and Bolton enrichment liquid were set as negative controls.

The results of the influence of the basic combination of the ICDC-CAMPY enrichment fluid and the Bolton enrichment fluid on the difference of the growth OD values of the campylobacter are shown in Table 1 and FIG. 1.

TABLE 1 differences in OD values of different enrichment solutions based on Campylobacter growth

As can be seen from the results of FIG. 1 and Table 1, the OD value of 1 time of bacterial growth was measured every 3h, and the results showed that the strains had consistent growth trends under different conditions, the OD values continued to increase after the first 12h and 15h in the logarithmic growth phase, and the strain entered the plateau phase after 24 h. However, the growth rate of the bacterial strain in the ICDC-CAMPY enrichment fluid is obviously higher than that of the Bolton enrichment fluid, and the growth peak value is higher.

Example 2 (verification of the Effect of different salt ion concentrations and addition of surfactants on Campylobacter growth)

(1) Taking a standard strain NCTC11168 of the campylobacter as a standard strain, carrying out passage on the standard strain NCTC11168 of the campylobacter in a Karmali culture medium, and carrying out microaerobic culture at 37 ℃ for 24 h;

(2) collecting the strains which grow vigorously, diluting the strains with sterile normal saline to the initial concentration of 0.05OD, respectively adding the strains into a culture medium which is formed by combining ICDC-CAMPY enrichment broth salt ions, growth promoting factors, antibiotics and surfactants and Bolton enrichment broth, and detecting the OD value of the growth of the strains after carrying out microaerobic culture for 24 hours.

On the basis of the components of the ICDC-CAMPY enrichment broth basic combination and the Bolton enrichment broth basic combination in the embodiment 1, different salt ion concentrations and other components such as surfactants, growth factors, antibiotics and the like are added, and the specific components and the experimental results are shown in a table 2 and a figure 2; wherein the surfactant is tween 800.2 mL; the growth factor is 50 parts of defibrinated rabbit blood; the antibiotics are 15mg of vancomycin, 15mg of amphotericin B15mg, 10mg of rifampicin, 10mg of trimethoprim and 30mg of cefoperazone sodium; the optimized metal salt concentration is 4g/L of sodium chloride, 2g/L of disodium hydrogen phosphate, 0.5g/L of sodium pyruvate, 0.5g/L of sodium metabisulfite and 0.25g/L of ferrous sulfate, and the unoptimized metal salt concentration is 4g/L of sodium chloride, 0.25g/L of sodium pyruvate and 0.25g/L of sodium metabisulfite.

TABLE 2 growth differences of different enrichment solutions for Campylobacter

As can be seen from Table 2 and FIG. 2, for the ICDC-CAMPY enrichment fluid, antibiotics are removed (the purpose of antibiotic addition is to selectively pollute the sample with infectious microbes), and other components can remarkably promote the growth of campylobacter.

Example 3 (analysis of the proliferation of the growth factor in an optimized salt ion in ICDC-CAMPY enrichment broth)

Based on example 2, the synergy analysis was performed for optimized salt ion concentration and growth factor addition in ICDC-camp y as follows:

(1) culturing Campylobacter standard strain ATCC33560 in Karmali solid culture medium for 16-20h, collecting strain in physiological saline, and repeatedly washing the strain;

(2) diluting the strain with normal saline, and adjusting the bacterial liquid to make the concentration of the bacterial liquid reach OD₆₀₀＝1；

(3) Will OD₆₀₀Diluting the bacterial liquid of which the ratio is 1 by 50 times by using physiological saline, and adding the diluted bacterial liquid into ICDC-CAMPY enriched bacterial liquid with the following different components respectively in the following sequence:

	different ICDC-CAMPY enrichment liquid components
		A1	Basal medium component + culture inhibitor and surfactant
A2	Basal medium component + culture inhibitor and surfactant + optimized salt ion
		A3	Basal medium component + culture inhibitor and surfactant + growth promoting factor
A4	Basal medium component, culture inhibitor, surfactant, optimized salt ion and growth promoting factor

Wherein the culture inhibitor is 1 part of sodium deoxycholate; the surfactant is 800.2 mL of Tween; the optimized metal salt concentration is 4g/L of sodium chloride, 2g/L of disodium hydrogen phosphate, 0.5g/L of sodium pyruvate, 0.5g/L of sodium metabisulfite and 0.25g/L of ferrous sulfate, and the unoptimized metal salt concentration is 4g/L of sodium chloride, 0.25g/L of sodium pyruvate and 0.25g/L of sodium metabisulfite; the somatomedin comprises 50 parts of defibrinated rabbit blood and 0.001 part of nicotinic acid.

(4) Culturing the culture solutions with different combinations in a microaerophilic environment at 42 deg.C for 30h, and measuring OD every 3h₆₀₀Values to determine the strain proliferation characteristics, mean was repeated three times for each combined sample.

The proliferation results of different combinations of enrichment strains at different time points are shown in table 3 and fig. 3.

TABLE 3 proliferation results (mean OD values) of different combinations of enrichment broth strains at different time points

	A1	A2	A3	A4
						3h	0.0572	0.0546	0.0466	0.0851
6h	0.157	0.1724	0.1987	0.3015
					9h	0.3114	0.3941	0.4344	0.6094
12h	0.5599	0.679	0.6857	0.8773
					15h	0.8193	0.9671	0.9341	1.1024
18h	1.0274	1.1156	1.1336	1.2444
					21h	1.212	1.2529	1.2994	1.3644
24h	1.3331	1.4041	1.4477	1.5354
					27h	1.4615	1.4334	1.3861	1.2955

As can be seen from table 3 and fig. 3, the basal medium component + culture inhibitor + surfactant + optimized salt ion + growth promoting factor (a4) of the ICDC-CAMPY enrichment broth significantly promoted the proliferation of campylobacter.

Examples 4-6 (differential verification of the different contamination samples ICDC-CAMPY enrichment broth culture and the general Bolton (Preston) enrichment broth culture)

Due to the complexity of food samples, isolated culture of campylobacter bacteria in food samples is difficult. The method comprises the steps of firstly determining that the background of a sample is free from campylobacter pollution from retail chicken samples which are most easily polluted and 3 important food samples such as milk and eggs which can cause outbreak in the world, preparing simulated pollution samples of target strains and other mixed strains of the sample (the pollution concentration of the mixed strains is 10 times of that of the target strains), and carrying out embodiment verification on the polluted samples by simultaneously detecting the combination of Bolton and Preston enrichment liquids and ICDC-CAMPY enrichment liquids. The method comprises the following specific steps:

1. preparation of simulated contamination strains:

(1) the target strains are campylobacter jejuni and campylobacter coli, and the information of the verified strains is shown in table 4:

TABLE 4 verification of background information of strains of interest

Serial number	Bacterial strain	Strain numbering	Origin of the Strain
				1	Campylobacter jejuni	SZ112	Chicken meat
2	Campylobacter jejuni	SZ113	Duck meat
				3	Campylobacter jejuni	SZ114	Chicken meat
4	Campylobacter jejuni	SZ115	Duck meat
				5	Campylobacter jejuni	SZ116	Chicken meat
6	Campylobacter coli	ATCC33559	Standard strains
				7	Campylobacter jejuni	ATCC33560	Standard strains
8	Campylobacter jejuni	ATCC700297	Standard strains
				9	Campylobacter jejuni	SZ-C064	Chicken anus wiper
10	Campylobacter jejuni	SZ-C065	Chicken anus wiper
				11	Campylobacter jejuni	SZ-C066	Cow dung
12	Campylobacter jejuni	BDYY-07267	Patient's health
				13	Campylobacter jejuni	BDYY-07270	Patient's health
14	Campylobacter coli	SH15CE11	Chicken manure
				15	Campylobacter coli	SH15CE12	Chicken manure
16	Campylobacter coli	SH15CE13	Chicken manure
				17	Campylobacter coli	SH-CCD11C353	Diarrhea patients
18	Campylobacter coli	SH-CCD11C361	Diarrhea patients
				19	Campylobacter coli	SH-CCD11C377	Diarrhea patients
20	Campylobacter coli	SZC102	Chicken liver

(2) The mixed bacteria mainly comprise proteus, salmonella, vibrio, escherichia coli, toxoplasma and the like, and the strain information is shown in table 5:

TABLE 5 verification of background information of the infectious microbe strains

Example 4: cultivation, detection and verification of campylobacter of pollution sample of raw poultry meat

(1) Preparing an ICDC-CAMPY enrichment liquid: weighing 10 parts of solid brain extract, 6 parts of bovine brain heart extract, 12 parts of peptone and 3 parts of glucose to form a nitrogen source and a carbon source; 4 parts of sodium chloride, 3 parts of disodium hydrogen phosphate, 0.25 part of sodium pyruvate, 0.25 part of sodium metabisulfite and 0.25 part of ferrous sulfate; 0.001 part of nicotinic acid; 0.5 part of deoxycholate sodium and 800.1 mL of Tween; dissolving the components in 1L sterile deionized water, cooling to about 50 ℃ after high pressure of 121 ℃ for 15 minutes, adding 50 parts of sterile defibrinated sheep blood, simultaneously adding 20mg of vancomycin, 15mg of amphotericin B, 10mg of rifampicin, 10mg of trimethoprim and 20mg of cefoperazone sodium to prepare ICDC-CAMPY enrichment broth, fully mixing the enrichment broth, subpackaging by 4 mL/tube, and refrigerating at 4 ℃ and keeping in the dark.

(2) Preparation of simulated raw poultry meat samples: respectively recovering and culturing target strains and mixed bacteria (the mixed bacteria are uniformly mixed into a bacteria liquid pool in the same amount of 1:1 by different strains), collecting the strains which grow vigorously in physiological saline, adjusting the bacteria liquid concentration of the campylobacter to be 1.0 McLeod, diluting by 10 times, taking 10uL to count (counting from 1: 10) (figure 4, left), and determining that the bacteria liquid concentration of the 1.0 McLeod is as follows: 2*10⁸CFU/mL, diluting the original campylobacter bacteria liquid by 10 times, wherein the diluted concentration is as follows: 2*10⁵，2*10⁴，2*10³， 2*10²，2*10¹、2*10⁰CFU/mL for a total of 6 contamination concentration gradients; adjusting the concentration of the mixed bacteria liquid pool to 2.5 McLeod, diluting 10 times, taking 10uL to count (figure 4. right), determining that the concentration of the mixed bacteria liquid with 2.5 McLeod is 1 x 10⁹CFU/mL, diluting the mixed bacteria liquid by 10 times, wherein the diluted concentration is as follows: 1*10⁶，1*10⁵，1*10⁴，1*10³，1*10²， 1*10¹CFU/mL, 6 contamination concentration gradients.

(3) And (3) verifying the culture of campylobacter coli and empty test of simulated live poultry meat samples by enrichment culture of different enrichment solutions:

1) bolton (preston) enrichment: directly carrying out Bolton (Preston) enrichment liquid microaerophilic enrichment culture according to a national standard method: respectively weighing 25g of chicken, putting the chicken into a homogeneous bag with a filter membrane, respectively adding 225mL of Bolton enrichment broth and 225mL of Preston (Preston) enrichment broth into the homogeneous bag, and fully washing; adding different concentrations of campylobacter bacteria liquid and mixed bacteria liquid (keeping the concentration of the mixed bacteria liquid and the target bacteria campylobacter bacteria liquid consistent or 10 times of the concentration of the target bacteria liquid) into the washing liquid, and mixing uniformly;

2) ICDC-CAMPY enrichment: adding 100mL of peptone water washing liquor into a quality bag for full washing, adding the polluted campylobacter and infectious microbes, fully and uniformly mixing, centrifuging the egg washing liquor by 2000g, and enriching and precipitating. Adding 0.4mL of the precipitation suspension into 4mL of ICDC-CAMPY enrichment broth, and performing microaerophilic enrichment culture with Bolton enrichment broth and Preston enrichment broth;

3) bolton (Preston) enrichment culture results (Bolton (Preston) enrichment culture results in the contamination concentrations of 6 campylobacter and infectious microbes in FIGS. 5, 1-6 respectively): pick 2 x 10⁶Concentration, 3 suspected monoclonals on a skerrow plate (24h, 1:50), and then 1 positive monoclone is determined through microscopic examination identification. Determining the concentration of Bolton enrichment broth and Preston enrichment broth to be 10 for the lowest detection limit of the polluted raw poultry meat sample⁵CFU/25 g. However, the colony of the target bacteria is similar to the colony of the miscellaneous bacteria in morphology and can be completed by an experienced operator.

4) The result of filtration culture after ICDC-CAMPY enrichment (FIGS. 6, 1-6 are enrichment culture results of 6 simulated campylobacter and mixed bacteria contamination concentration samples, respectively): selecting colonies from the lowest pollution concentration, identifying campylobacter, and determining the concentration to be 2 x 10⁰CFU/25g is the minimum detection concentration (i.e., the detection limit of the experiment) for the enrichment culture of ICDC-CAMPY. The enrichment culture comparison results of different enrichment liquids confirm that ICDC-CAMPY is high-efficiency campylobacter enrichment liquid for the polluted raw poultry meat.

In conclusion, the experimental results of this example confirmed that the minimum detected concentration of ICDC-CAMPY enrichment fluid for contaminated raw poultry meat was 2 x 10⁰CFU/25g。

Example 5: culture, detection and verification of campylobacter of milk pollution sample

(1) Preparing ICDC-CAMPY enrichment liquid: weighing a nitrogen source and a carbon source which are composed of 12 parts of solid brain immersion liquid, 6.5 parts of bovine brain heart extract, 10 parts of peptone and 2 parts of glucose; 4 parts of sodium chloride, 2 parts of disodium hydrogen phosphate, 0.5 part of sodium pyruvate, 0.5 part of sodium metabisulfite and 0.25 part of ferrous sulfate; 0.001 part of nicotinic acid; 0.5 part of deoxycholate sodium and 0.2mL of Tween 80; dissolving the components in 1L sterile deionized water, cooling to about 50 ℃ after high pressure at 121 ℃ for 15 minutes, adding 50 parts of sterile defibrinated rabbit blood, simultaneously adding 15mg of vancomycin, 15mg of amphotericin B15mg, 10mg of rifampicin, 10mg of trimethoprim and 25mg of cefoperazone sodium to prepare ICDC-CAMPY enrichment broth, fully mixing the enrichment broth, subpackaging with 4 mL/tube, refrigerating at 4 ℃ and keeping in the dark.

(2) Preparation of a simulated pollution sample:

the preparation method of the campylobacter and the mixed bacteria is consistent with the preparation method, and the pollution concentration of the campylobacter and the mixed bacteria in 50mL of milk is as follows in sequence: 3*10⁴CFU、3*10³CFU、3*10²CFU、3*10¹CFU、3*10⁰CFU、3*10^-1A CFU; mixed bacteria: 3*10⁵CFU、 3*10⁴CFU、3*10³CFU、3*10²CFU、3*10¹CFU、3*10⁰And (4) CFU. Respectively at 6 concentrations.

(3) Culturing different enrichment liquids:

1) respectively taking 50mL of liquid milk, respectively adding the prepared campylobacter and infectious microbe bacterial liquid into each sample, and fully and uniformly mixing;

2) bolton (preston) enrichment: centrifuging the above contaminated sample at 20000g for 30min according to the national standard method, discarding the supernatant, respectively suspending and precipitating with 10mL Bolton (Preston) enrichment broth (to avoid bringing into oil layer as much as possible), transferring to 90mL Bolton (Preston) enrichment broth, and performing microaerophilic culture;

3) ICDC-CAMPY enrichment: suspending with 1mL of physiological saline, adding 400uL of the suspension into a screwed pipe containing 4mL of ICDC-CAMPY enrichment medium, and performing microaerophilic enrichment culture;

4) the results after Bolton (Preston) enrichment are shown in FIG. 7, 6 pollution concentrations are respectively shown from 1 to 6, a plate colony is picked, campylobacter is not found after identification, therefore, the Bolton (Preston) enrichment is applied to culture campylobacter in polluted milk, and the detection limit is larger than 3 x 10⁴CFU/50mL。

5) The enrichment culture results of ICDC-CAMPY are shown in FIG. 8, 6 contamination concentrations from 1-6 are respectively, the plate colonies are picked, the single colony on the plate with the lowest concentration is identified as campylobacter (4 th concentration), and the concentration is determined to be 3 x 10¹CFU/50mL is the minimum detection concentration (i.e., detection limit of the experiment) of the contaminated milk sample and ICDC-CAMPY enrichment culture.

Example 6: culture detection verification of campylobacter of egg white pollution sample

(1) Preparing an ICDC-CAMPY enrichment liquid: weighing a nitrogen source and a carbon source which are composed of 13 parts of solid brain immersion liquid, 6 parts of bovine brain heart extract, 10 parts of peptone and 2 parts of glucose; 4 parts of sodium chloride, 2.5 parts of disodium hydrogen phosphate, 0.3 part of sodium pyruvate, 0.3 part of sodium metabisulfite and 0.25 part of ferrous sulfate; 0.001 part of nicotinic acid; 1.0 part of deoxysodium cholate and 0.1mL of Tween 80; the components are dissolved in 1L sterile deionized water, the mixture is cooled to about 50 ℃ after being pressurized at 121 ℃ for 15 minutes, 50 parts of sterile fetal calf serum is added, 20mg of vancomycin, 15mg of amphotericin B, 10mg of rifampicin, 20mg of trimethoprim and 30mg of cefoperazone sodium are added at the same time to prepare ICDC-CAMPY enrichment broth, the enrichment broth is fully mixed and then packaged in 4mL tubes, and the mixture is refrigerated at 4 ℃ and stored in dark place.

(2) Preparing an egg white polluted sample: the preparation method of the campylobacter and the mixed bacterium is the same as that of the campylobacter and the mixed bacterium; taking 25mL egg white sample to 125mL Bolton enrichment broth (diluted 1: 6), adding diluted campylobacter and mixed bacteria broth respectively to obtain campylobacter 1.5 x 10⁷CFU、1.5*10⁶CFU、1.5*10⁵CFU、1.5*10⁴CFU、1.5*10³CFU、1.5*10²CFU6 dilutions and 1.5 x 10⁸CFU、1.5*10⁷CFU、1.5*10⁶CFU、1.5*10⁵CFU、1.5*10⁴CFU、1.5*10³Mixed solution of CFU mixed bacteria with 6 dilutions.

(3) Culturing the polluted samples by different enrichment liquids:

1) bolton (preston) enrichment: adding the mixture into Bolton (Preston) enrichment liquid, uniformly mixing, transferring 25mL of the mixture into 100mL of Bolton (Preston) enrichment liquid, uniformly mixing, and performing microaerophilic enrichment culture on the Bolton broth after enrichment;

2) ICDC-CAMPY enrichment: taking 25mL of egg white to dilute and mix evenly in 75mL of 0.1% sterile peptone water washing liquid, and respectively adding 6 dilutions of the diluted campylobacter bacteria liquid and the mixed bacteria liquid into each sample; 2000g peptone water washing liquor centrifugally polluted at 4 ℃ for 15min, discarding supernatant, carrying out suspension precipitation by using 1mL physiological saline, and carrying out microaerophilic enrichment culture on 0.4mL precipitation suspension liquid in 4mL ICDC-CAMP enrichment broth;

3) the results of the culture after the Bolton (Preston) enrichment are shown in figure 9, 6 pollution concentrations are respectively obtained from 1 to 6, the plate bacterial colonies are picked, no campylobacter is found after identification, therefore, the judgment that the campylobacter in the polluted egg white is cultured by applying the Bolton (Preston) enrichment is that the detection limit is more than 1.5 x 10⁷CFU/25mL。

4) The culture result after enrichment of ICDC-CAMPY enrichment liquid is shown in figure 10, the contamination concentration is 6 from 1 to 6, the plate bacterial colony is picked up, the single bacterial colony on the plate with the lowest concentration is identified to be campylobacter, and the concentration is determined to be 2 x 10¹cfu/25mL is the minimum detection concentration (i.e., detection limit of the experiment) of the contaminated egg white sample ICDC-CAMPY enrichment culture.

To sum up, the embodiment of the application optimizes the composition of the enrichment fluid, modifies the components of the basic culture medium, removes the nitrogen sources and the carbon sources such as meat peptone, albumin and hydrolysate of complex protein and yeast extract in the general basic culture medium based on the Bloton enrichment fluid, and changes the solid bovine brain heart extract, peptone, glucose and nicotinic acid which have the promotion effect on fastidious bacteria, so as to obviously improve the growth speed of microaerophilic campylobacter, and can obtain the culture and single colony of the campylobacter in the detection of the sample simultaneously polluted by the campylobacter and the infectious microbes.

Secondly, by adding surfactants of sodium deoxycholate and tween, the growth of campylobacter is promoted, other mixed bacteria are inhibited, and the effect of the bacteria increasing liquid under the standing condition is not obviously different from that of vibration bacteria increasing; and by changing the components and the concentration of metal ions in the enrichment liquid, the use of sodium pyruvate, sodium metabisulfite and ferrous sulfate in the enrichment liquid is increased, the concentration of part of ions is improved, and the growth of campylobacter is promoted.

Moreover, the ICDC-CAMPY component combination provided by the embodiment of the application is beneficial to the proliferation of campylobacter, improves the movement capacity of the 'viable bacteria' of campylobacter trophotrophicus, and greatly improves the detection limit of campylobacter in a sample from 10 times⁴CFU-10⁷CFU to 10⁰CFU; in addition, the application is implemented inThe use of enrichment liquid is reduced in the use process: the peptone water wash sample or the liquid sample is directly centrifuged, the ICDC-CAMP enriched liquid is applied after enrichment, only a small amount of enriched sample is needed in the sample separation culture, and at least 225mL of Bolton or Preston enriched liquid needed by 25g of poultry meat is needed according to the method of the national standard, but only 4mL of ICDC-CAMP enriched liquid is needed.

It is worth noting that only the sensitivity of molecular detection can be compared with the enrichment culture method, but no other methods with the same sensitivity and specificity can be replaced at present in the enrichment culture method because the molecular detection cannot obtain 'living' bacteria.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and scope of the present application should be included in the present application.

Claims

1. A campylobacter enrichment culture solution is characterized by comprising the following raw materials in parts by weight:

10-15 parts of brain extract, 6-7 parts of bovine brain heart extract, 8-12 parts of peptone, 1-3 parts of glucose, 6.5-12.5 parts of metal salt, 50 parts of blood, 0.001-0.002 part of nicotinic acid, 0.5-1.5 parts of deoxysodium cholate, 0.0001-0.0005 part of tween and 0.05-0.1 part of antibiotic.

2. The campylobacter enrichment culture solution of claim 1, wherein the metal salt comprises the following raw materials in parts by weight:

3. The campylobacter enrichment culture medium of claim 1, wherein the blood is one of defibrinated sheep blood, rabbit blood, and fetal calf serum.

4. The campylobacter enrichment culture solution of claim 1, wherein the antibiotics comprise the following raw materials in parts by weight:

0.01-0.02 part of vancomycin, 0.01-0.015 part of amphotericin B, 0.01-0.012 part of rifampicin, 0.01-0.015 part of trimethoprim and 0.02-0.032 part of cefoperazone sodium.

5. A preparation method of a campylobacter enrichment culture solution is characterized by comprising the following steps:

weighing the raw materials according to the formula of the campylobacter enrichment culture solution of any one of claims 1-4 for later use;

6. The method for preparing the enriched culture solution of campylobacter according to claim 5, wherein said brain infusion, bovine brain extract, peptone, glucose, metal salts, nicotinic acid, sodium deoxycholate and tween are dissolved in deionized water, and subjected to high pressure treatment to obtain a mixed solution, comprising:

7. The method of claim 5, wherein the step of adding blood and antibiotics to the mixture is performed to produce the Campylobacter enrichment culture, comprising:

8. Use of a campylobacter enrichment culture of any one of claims 1-4 for campylobacter detection.